Polynucleotides encoding MHC class I-restricted hTERT epitopes, analogues thereof or polyepitopes

ABSTRACT

This invention relates to the field of anticancer therapy, and to the identification of immunogenic peptides derived from the human telomerase reverse transcriptase (hTERT). The present invention relates to polynucleotides encoding hTERT epitopes restricted to MHC class I molecule, analogs thereof and polyepitopes containing such epitopes and/or analogs. Are also included in the present invention, vector and cell comprising such polynucleotides. The present invention also concerns composition comprising hTERT polypeptides, corresponding polynucleotides, vectors and cells, for use in the treatment and/or prevention of cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry of PCT/EP2006/007571, filedJul. 31, 2006, which claimed priority of European Patent Application No.05291627.7 filed Jul. 29, 2005.

This invention relates to the field of anticancer therapy, and to theidentification of immunogenic peptides derived from the human telomerasereverse transcriptase (hTERT). The present invention relates topolynucleotides encoding hTERT epitopes restricted to MHC class Imolecule, analogues thereof and polyepitopes containing such epitopesand/or analogues. Are also included in the present invention, vectorsand cells comprising such polynucleotides. The present invention alsoconcerns compositions comprising hTERT polypeptides, correspondingpolynucleotides, vectors and cells, for use in the treatment and/orprevention of cancer.

In light of deficits in current anticancer therapeutic approaches,antitumor therapy has enjoyed renewed interest. Recent studies haveenhanced our understanding of anti melanoma immune responses associatedwith tumor regression. Collectively these data suggest that activatedtumor-specific CD8 CTL are immunological weapon of choice for potentanti-tumor therapy. The research of over-expressed proteins permittingto trigger cytotoxic T lymphocytes to tumours of different origins isalso progressing.

Telomerase is a ribonucleoprotein complex, consisting of a proteincomponent, TERT, and an RNA component (TR) containing the template forthe synthesis of the repeat unit (T₂AG₃) added onto the ends ofchromosomes, that stabilizes the chromosomes during replication andprevent end-to-end fusion. Maintenance of a constant telomere lengthprevents cells from aging, and confers immortality (Hahn et al. Nat Med1999; 5:1164-70). High hTERT activity was found in more than 85% ofhuman cancers, whereas most normal adult human tissues show no or littletelomerase activity (Counter et al. Blood 1995; 85:2315-20).

The widespread expression of telomerase in tumors indicates that peptidefragments of hTERT could serve as tumor specific antigen(s) and this hasbeen confirmed in several reports (Vonderheide et al. Immunity 1999;10:673-9). Recent data from Phase I clinical trials demonstrate thefeasibility of vaccine against hTERT in HLA-A2⁺ patients, opening theway for use of hTERT for therapeutic vaccination (Vonderheide et al.Clin Cancer Res 2004; 10:828-39; Parkhurst et al. Clin Cancer Res 2004;10:4688-98). Nevertheless, the immunogenic hTERT peptides identified todate are restricted to one MHC allele HLA-A2.1, with only two initialreports on two HLA-supertypes, HLA-A3 and HLA-A24, representedrespectively in 44.2% and 40% of the population.

Hence, the following publications reported the identification of hTRETpeptides:

-   -   hTERT peptide ILAKFLHWL (SEQ ID NO: 1), restricted to HLA-A2        (Vonderheide et al. Immunity 1999; 10:673-9),    -   hTERT peptides MPRAPRCRA (SEQ ID NO: 2), RPAEEATSL (SEQ ID NO:        6), RPSFLLSSL (SEQ ID NO: 10) and APRCRA VRS (SEQ ID NO: 2)        identified by informatics prediction within the HLA-A context WO        00/02581). However, these peptides have never been confirmed by        experimental results to be efficient epitopes, neither in a        HLA-87 context nor in any HLA context.    -   hTERT peptide KLFGVLRLK (SEQ ID NO: 3) (K973), restricted to        HLA-A3 (Vonderheide et al. Clin Cancer Res 2001; 7:3343-8), and    -   hTERT peptides VYAETKHFL (SEQ ID NO: 4) (TEL324) and VYGFVRACL        (SEQ ID NO: 5) (TEL 461), restricted to HLA-A24 (Arai et al.        Blood 2001; 9:2903-7)

Consequently, the hTERT peptides identified so far do not cover all thepopulation, thus excluding a large segment of patients.

In order to overcome this failure, at least in part and to thus bettercover the genetic diversity of the human population, the presentinvention identifies new epitopes derived from hTERT, restricted to aparticular HLA which is different from HLA-A3 and HLA-A24. Among themany different alleles, the present application is interested in theHLA-B7 supertype which is expressed in about 25% of the population, andparticularly to the second allele the most expressed in the human HLA-Bpopulation: the HLA-*B0702 (allele present in 15-20% of individuals inhuman population).

The gene of the isoform-1 of the telomerase is 4015 base pairs (bp) long(NCBI Accession number AF015950) and encodes a protein of 1132 aminoacids (NCBI Accession number AAC51672.1) (FIG. 1).

The invention concerns a polynucleotide encoding a human telomerasereverse transcriptase (hTERT) peptide. In a particular embodiment of theinvention, the encoded peptides are 9 amino acids in length (nonamer) or10 amino acids in length (decamer), and the polynucleotide has hence 27or 30 nucleotides. In general, the encoded peptide is less than 15 aminoacids and the polynucleotide has less than 45 nucleotides.

The invention also concerns a polynucleotide encoding hTERT peptidesthat are epitopes, restricted to MHC class I molecule, especiallyepitopes suitable to induce an immune response restricted to HLA-87. Thenucleotide sequence of the polynucleotide of the invention is, in aparticular embodiment, limited to the sequence encoding the hTERTpeptide. Such peptides can be chosen from the group consisting ofMPRAPRCRA (SEQ ID NO: 6) (p1; amino acid residues 1 to 9), APRCRAVRSL(SEQ ID NO: 7) (p4; amino acid residues 4 to 13), APSFRQVSCL (SEQ ID NO:8) (p68; amino acid residues 68 to 77), RPAEEATSL (SEQ ID NO: 9) (p277;amino acid residues 277 to 285), RPSFLLSSL (SEQ ID NO: 10) (p342; aminoacid residues 342 to 350), RPSLTGARRL (SEQ ID NO: 11) (p351; amino acidresidues 351 to 360), DPRRLVQLL (SEQ ID NO: 12) (p444, amino acidresidues 444 to 452), FVRACLRRL (SEQ ID NO: 13) (p464, amino acidresidues 464 to 472), AGRNMRRKL (SEQ ID NO: 141 (p966, amino acidresidues 966 to 974), LPGTTLTAL (SEQ ID NO: 15) (p1107, amino acidresidues 1107 to 1115) and LPSPKFTIL (SEQ ID NO: 16) (p1123, amino acidresidues 1123 to 1131). All these polynucleotides may be used to inducea HLA-87-restricted immune response. In a particular embodiment, theinvention especially concerns a polynucleotide encoding aHLA-87-restricted hTERT epitope, chosen from the group consisting ofRPSL TGARRL (SEQ ID NO: 11) (p351), APSFRQVSCL (SEQ ID NO: 8) (p68),APRCRAVRSL (SEQ ID NO: 7) (p4), DPRRLVQLL (SEQ ID NO: 12) (p444),FVRACLRRL (SEQ ID NO: 13) (p464), AGRNMRRKL (SEQ ID NO: 14) (p966),LPGTTL TAL (SEQ ID NO: 15) (p1107) and h LPSPKFTIL (SEQ ID NO: 16)(p1123).

As defined herein, an “epitope” is an antigenic determinant, i.e. thepeptide site recognized by cells of the immune system (immune cells) andespecially the site necessary to elicit an immune response. The termepitope encompasses both linear epitope for which the consecutive aminoacids (especially, 9 or 10) are recognized by immune cells and,conformational epitope for which immune cells recognize amino acids tothe extent they adopt a proper configuration or conformation.Consequently, in some epitopes, the conformation (three dimensionalstructure) is as important as the amino acid sequence (primarystructure).

The expression “MHC class I-restricted” refers to the capacity for aparticular peptide or epitope to have an affinity for a MHC (majorhistocompatibility complex) molecule of class I. Similarly; theexpression “HLA-B7-restricted” refers to the capacity for a particularpeptide or epitope to have an affinity for this type of HLA molecule.

Briefly, MHC genes encode cell surface polymorphic molecules that do notbind only foreign peptides but also can bind overexpressed or not selfpeptides or mutated self peptides, to display them on the cell surfaceof cell enabling their recognition by appropriate immune cells,especially T-cells. Said MHC molecules, referred to as H-2 in mice andHLA (Human Leucocyte Antigen) in humans, are classified as either classI molecules (designated HLA-A, B, or C) or class II molecules(designated DP, DQ or DR).

Accordingly, MHC class I molecules specifically bind CD8 moleculesexpressed on cytotoxic T lymphocytes (also named TCD8⁺), whereas MHCclass II molecules specifically bind CD4 molecules expressed on helper Tlymphocytes (TCD4⁺).

MHC class I molecules bind peptides derived from proteolyticallydegraded proteins especially endogenously synthesized proteins, by acell. Small peptides obtained accordingly are transported into theendoplasmic reticulum where they associate with nascent MHC class Imolecules before being routed through the Golgi apparatus and displayedon the cell surface for recognition by cytotoxic T lymphocytes.

In the present invention, the above-identified peptides have been shownon the one hand to bind either with high or medium affinity to MHC classI molecule, and on the other hand to be efficiently transported as aMHC/epitope complex to the cell surface of cells. In a preferredembodiment, the MHC class I molecule is an MHC allele of the HLA-B7supertype family; the hTERT epitope is said HLA-B7 supertype-restricted.Said family encompasses alleles B0702, B0703, B0704, B0705, B1508,B3501, B3502, B3503, B51, B5301, B5401, B5501, B5502, B5601, B5602,B6701 and B7801, family from which the HLA-B0702 is preferred(HLA-B0702-restricted hTERT epitope).

A MHC stabilization assay may be used to test the affinity of a peptidefor a particular HLA class I molecule (relative avidity), such as theone described in Firat et al. (1999. Eur. J. of Immunol. 29: 3112-3121),incorporated herein by reference. Briefly, MHC class Imolecule-transfected cells are incubated overnight at 2×10⁵ cells/wellin 96-well plates in serum free medium AIM-V (Invitrogen Corp., Gibco),supplemented with 100 ng/ml of human β2-microglobulin,

-   -   in the absence of peptides (negative control),    -   in the presence of a reference peptide (positive control), and    -   in the presence of the peptides to be tested (hTERT peptides in        the present case).        Peptides are incubated at various final concentrations ranging        from 0.1 to 100 μM (with intermediate concentrations of 1 and 10        μM). The transfected cells are then labelled with a saturating        concentration of an antibody recognizing the particular HLA MHC        class I molecule, then washed twice and finally stained with a        secondary antibody before flow cytometry. Results are expressed        as values of relative avidity, that is the ratio of        concentration of tested peptide necessary to reach 20% of the        maximal binding by the reference to that of the reference        peptide. Therefore, the lower the value, the stronger the        binding. Following this method, a peptide is said to have a high        relative affinity for a particular HLA class I molecule, when        RA<1. In contrast, a medium relative affinity is concluded when        RA is comprised between 1 and 5, and preferably between 1 and 3.

Among the hTERT nonamers and decamers, and particularly theabove-identified peptides, the following MHC class I-restricted hTERTepitopes can be classified as having high relative affinity for MHCclass I molecule: MPRAPRCRA (SEQ ID NO: 6 (p1), APRCRAVRSL (SEQ ID NO:7) (p4) and APSFRQVSCL (SEQ ID NO: 81 (p68). Using the same approach,the following MHC class I-restricted hTERT epitopes can be classified ashaving a medium relative affinity for MHC class I molecule: RPAEEATSL(SEQ ID NO: 9) (p277), RPSFLLSSL (SEQ ID NO: 10) (p342), DPRRL VQLL (SEQID NO: 12) (p444) and RPSLTGARRL (SEQ ID NO: 11) (p351).

The present invention also relates to a polynucleotide encoding a MHCclass I-restricted epitope analogue, i.e., epitopes having at least oneamino acid substitution compared to a class I-restricted hTERT epitopeas described above, especially HLA-B7-restricted epitope analogue.

The term “analogue” as defined herein relates to a peptide whosesequence is derived from a hTERT peptide as described above by at leastone amino acid substitution, either conservative, semi-conservative ornon-conservative. An analogue is opposed to an epitope by the fact thatits nucleotide and/or amino acid sequence is not found in the referencehTERT gene or protein disclosed in FIG. 1 which are considered withinthe present application as the molecules of reference to define theso-called wild type peptides or polynucleotides. Such an analogue mayresult from the substitution in the corresponding nucleotide sequence ofone or several nucleic base(s) in the codon encoding said amino acid.Therefore, a polynucleotide analogue differs from its wild typecounterpart (polynucleotide encoding hTERT peptide with the referencesequence from which the peptide analogue is derived from) by at leastone substitution, and preferably one, two or three substitutions in thecodon(s) encoding the amino acid residue to be substituted. As anexample, the APRRLVQLL (SEQ ID NO: 17) peptide (called p444*) is derivedfrom the p444 peptide by the substitution of the first amino acidresidue (D→A).

A particular analogue of a HLA-B7-restricted analogue has the samelength or is shorter than the epitope from which it derives.

As a particular embodiment, the hTERT epitopes described above or theanalogues always conserve in their primary structure a proline (P) inposition 2, and/or one of the following amino acids in the lastC-terminal position: A, L, I, M, V, F, W or Y. Therefore, the hTERTpeptides, including the analogues, have the following consensussequence: X-P-X₆₋₇-[ALIMVFWY], wherein X refer to any amino acid, X₆₋₇refers to the number of amino acids and [ALIMVFWY] refers to one ofthese amino acids.

Therefore, to provide an epitope analogue, the amino acid substitutionor the corresponding codon substitution in the polynucleotide is notlocated in the second position (or second codon). In a preferredembodiment, no substitution is carried out in the C-terminal position,even though the last C-terminal amino acid can be replaced by anequivalent amino acid, i.e. either A, L, I, M, V, F, W or Y. Finally, ina preferred embodiment, the substitution is located in the first aminoacid position, wherein any amino acid is replaced by an alanine (A).

In a further embodiment of the invention, the last C-terminal amino acidof a decamer is deleted to give a nonamer, provided that the resultingnonamer maintains or adopts the X-P-X₆₋₇-[ALIMVFWY] consensus sequence.In the same way, an amino acid selecting among A, L, I, M, V, F, W and Yis added at the C-terminal end of a nonamer to give rise to decamer,provided that the resulting decamer maintains or adopts theX-P-X₆₋₇-[ALIMVFWY] consensus sequence.

In case of substitution, deletion or addition, including especiallythose illustrated above, taken individually or as combinations, thetridimensional conformation of the peptide analogue must be the same orslightly modified with respect to the one of the wild type counterpart,to ensure a correct folding of the analogue and its correct binding tothe MHC class I molecule. The MHC stabilization assay described abovecan be used to check that such constraints are fulfilled.

The resulting analogue has at least the same characteristics as its wildtype counterpart, in terms of affinity for a particular MHC class Imolecule, especially HLA-B7 molecule, and has essentially the samecapacity to be transported as an epitope/MHC complex on the cell surfaceand/or has essentially the same capacity to elicit an immunogenicresponse when tested in the same conditions.

In a preferred embodiment, the starting peptide is a hTERT epitopehaving a medium affinity, and the resulting analogue has a higheraffinity than its wild type counterpart. In another embodiment, theanalogue has a higher immunogenicity than its wild type counterpart. Asan example, the p444* peptide analogue quoted above has an increasedaffinity for MHC class I molecule compared to the p444 peptide fromwhich it is derived from.

This is an object of the invention to provide hTERT epitope or analogue,able to elicit an immune response, and particularly a CTL response(Cytotoxic T Lymphocyte). However, the T lymphocytes do not recognizesaid analogue that is used to stimulate said lymphocytes only, viaantigen presenting cells. In an embodiment of the invention, theanalogue as described above keeps its immunogenic behaviour, and is ableto elicit an immune response against cells overexpressing hTERTepitopes, i.e. that CTLs recognize the wild type epitope, even ifstimulated with an epitope analogue. In a particular embodiment, thelymphocytes stimulated by an epitope analogue of the invention do notreact against cells, which do not overexpressed hTERT epitopes.Therefore, stimulated lymphocytes do not react with cells overexpressingother epitopes (cross reaction) or with cells expressing hTERT epitopeas basal level (healthy cells). In a particular embodiment, all thecharacteristics mentioned above are in a HLA-B7 environment, andpreferably in a HLA-B0702 context.

A conventional cytotoxicity assay may be performed by using a standard4-5 h ⁵¹Cr release assay to test the capacity of stimulated lymphocytesto react against target cells, such as in the Firat publication (1999.Eur. J. of Immunol. 29: 3112-3121) incorporated herein by reference.Briefly, cell suspension containing CTLs, are activated with peptide(hTERT peptide or analogue in the present case) plus self MHC Class Imolecule in vivo. Target cells (expressing or not the correspondingHTERT epitope) previously incubated with ⁵¹Cr, are then incubated withactivated lymphocytes. The recognition of target cells by activated CTLleads to the apoptosis of target cells and the release of ⁵¹Cr, whereinsaid release is proportional to the number of target cells killed. Anincubation of target cells with a control peptide is used as a negativecontrol to calculate the spontaneous release of ⁵¹Cr. Specificpercentage of lysis is calculated by subtracting non-specific lysisobserved with the control peptide to lysis obtained with the peptides tobe tested. The higher the percentage, the more targets have been killedby the CTL. Specific lysis is determined at several ratios of Effector(CTL) to target cells (E:T). The specific lysis is calculated as theratio between [experimental release−spontaneous release] and [totalrelease−spontaneous release].

The present invention also concerns a polynucleotide encoding apolyepitope. A polyepitope is defined as a polypeptide having at leasttwo epitopes, chosen among the MHC class I-restricted, especiallyHLA-B7-restricted hTERT epitopes (p1, p4, p68, p277, p342, p351, p444,p464, p966, p1107 and p1123) and/or MHC class I-restricted, especiallyHLA-B7-restricted, epitope analogues of the invention. Thepolynucleotide of the invention comprises or consists of at least twopolynucleotide units encoding said epitopes or analogues. Apolynucleotide unit is defined as the coding sequence for an epitope oranalogue of the invention as disclosed herein.

The invention particularly concerns a polynucleotide encoding apolyepitope, comprising at least two epitopes chosen among (a)RPSLTGARRL (SEQ ID NO: 11) (p351), (b) APSFRQVSCL (SEQ ID NO: 8) (p68),(c) APRCRAVRSL (SEQ ID NO: 7) (p4), (d) DPRRLVQLL (SEQ ID NO: 12)(p444), (e) FVRACLRRL (SEQ ID NO: 13) (p464), (f) AGRNMRRKL (SEQ ID NO:14) (p966), (g) LPGTTLTAL (SEQ ID NO: 15) (p1107) and (h) LPSPKFTIL (SEQID NO: 16) (p1123), or their analogues as defined above. Anotherpolynucleotide, encoding a polyepitope, comprises at least onepolynucleotidic unit chosen in the group of either a polynucleotideencoding a HLA-B7-restricted hTERT epitope corresponding to (a)RPSLTGARRL (SEQ ID NO: 11) (p351), (b) APSFRQVSCL (SEQ ID NO: 8) (p68),(c) APRCRAVRSL (SEQ ID NO: 7) (p4), (d) DPRRLVQLL (SEQ ID NO: 12)(p444), (e) FVRACLRRL (SEQ ID NO: 13) (p464), (f) AGRNMRRKL (SEQ ID NO:14) (p966), (g) LPGTTLTAL (SEQ ID NO: 15) (p1107) and (h) LPSPKFTIL (SEQID NO: 16) (p1123) and/or their analogues as defined above, and at leastone polynucleotidic unit chosen in the group of the polynucleotidesencoding the sequence MPRAPRCRA (SEQ ID NO: 6) (p1), RPAEEATSL (SEQ IDNO: 9) (p277) or RPSFLLSSL (SEQ 10 NO: 10) (p342), or their analogues asdefined above.

None of the polyepitope-encoding polynucleotides of the inventioncoincides with the coding sequence of the full length hTERT.

In a particular embodiment of the invention, the number of MHC class Irestricted hTERT epitopes and/or analogues in the prepared polyepitopeis limited to 30. In another embodiment, the number of HLA-B7 restrictedhTERT epitopes and/or analogues is limited to approximately 30, and ispreferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30. In anotherembodiment, the number of HLA-B0702 restricted hTERT epitopes and/oranalogues is limited to approximately 10, and is preferably 2, 3, 4, 5,6, 7, 8, 9 or 10.

Accordingly, the polynucleotide encoding a polyepitope has 30 or lesspolynucleotide units, especially 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25or 30 or any number within this range.

In a particular embodiment the polynucleotide units (and accordingly theepitopes) of the polynucleotide are consecutive.

In such a particular embodiment of the invention, the size of thenucleic sequence encoding the consecutive hTERT epitopes or analogues isless than 3000 bp, and preferably less than 2000 bp, 1000 bp, 500 bp,400 bp, 300 bp, 200 bp or 100 bp.

In a particular embodiment, the polynucleotide encoding the multipleepitopes (polyepitope) consists of a nucleic acid molecule encoding atruncated or mutated form of the hTERT protein. In a preferredembodiment, the truncated or mutated form of the hTERT protein isdeprived of its catalytic activity, i.e., is not capable to direct thesynthesis of the repeat unit (T₂AG₃) at the ends of chromosomes,participating in the maintenance of telomere length. Such a hTERTprotein deprived of its catalytic activity i.e., the retrotranscriptaseactivity, is said non-functional. Therefore, in another particularembodiment, the nucleic acid molecule encoding a truncated or mutatedform of the hTERT protein lacks the catalytic activity domain of hTERT.In a particular embodiment, the nucleic acid molecule encoding atruncated form of the hTERT protein encodes a protein consisting of atleast the 500 last C-terminal amino acids.

In a particular embodiment, the polynucleotide encodes a hTERT proteinthat is deleted for amino acids 867 to 869 (VDD sequence), correspondingto nucleotides 2654 to 2662 of FIG. 1 (wild-type) and providingnucleotide 2657 to be contiguous to nucleotide 2658 in FIG. 9representing the deletion site, or alternatively for amino acids 864 to872, corresponding to nucleotides 2645 to 2671 of FIG. 1 (wild-type) andproviding nucleotide 2648 to be contiguous to nucleotide 2649 in FIG. 10representing the deletion site. In a particular embodiment, the encodedhTERT protein has a deletion that comprises at least the amino acidresidues 867 to 869 i.e., that the deletion is larger than the 3 aminoacid residues (VDD sequence). As an example are the 864-872 deletiondescribed above as well as a 22 amino acid deletion starting from aminoacid residue 857 to 879 (according to FIG. 1) or a deletion comprisingthe 5 amino acids N-terminal and the 5 amino acids C-terminal to the VDDsequence (from amino acid 862 to amino acid 874 according to FIG. 1,corresponding to nucleotides 2639 to 2679). In a particular embodiment,the invention concerns a polynucleotide comprising or consisting of thenucleotide sequence as set forth in FIG. 9 or FIG. 10.

The polynucleotide units encoding the multiple epitopes of the inventioncan be arranged consecutively, i.e., the 3′ end of the firstpolynucleotide unit is directly linked to the 5′ end of the secondpolynucleotide unit (and so on), resulting in a polynucleotide encodinga peptidic sequence exclusively composed of consecutive epitopes. Such apolynucleotide can encode a polyepitope comprising or consisting of thep1, p4, p68, p277, p342, p351, p444, p464, p966, p1107 and p1123peptides. In a particular embodiment, the polynucleotide encodes thefollowing peptidic sequenceMPRAPRCRAAPRCRAVRSLAPSFRQVSCLRPAEEATSLRPSFLLSSLRPSLTGARRL (SEQ ID NO:20), comprising thus 6 MHC class I-restricted, particularly HLA-B7restricted, hTERT epitopes.

The multiple epitopes of the invention can alternatively be separated bya one-amino acid spacer or a peptide spacer, i.e., meaning that thedifferent polynucleotide units are separated by one or several codon(s)encoding respectively one or several amino acid(s). As spacers improvingthe processing of multiple epitopes, 4 amino acid-peptides composed ofan arginine (R) in the C terminal position and hydrophilic residues (A,K, D and/or T) in other positions are preferred. Especially, 4 aminoacid-peptides having a positively charged residue or an acidic residuein the C terminal position may be used, dependently or independently ofhydrophilic residues (A, K, D and/or T) in other positions. In aparticular embodiment, said spacers are internal processing sequencessuch as endosomal or lysosomal processing sequences, enabling the betterprocessing of the multiple epitopes and avoiding the processing of newpeptides resulting from overlapping cutting. Such a separation havingrecourse to a spacer can be used to separate all or, to the contrary,part of the polynucleotide units and accordingly, all or part of theepitopes.

The order in which the epitopes are arranged can be determined by theman skilled in the art, according to the following criteria: some ordersmay facilitate either the transcription and/or the translation of thepolynucleotide, may facilitate the transport of the resulting expressedpolyepitope in the endoplasmic reticulum (ER), especially if thetridimensional conformation impacts the properties, and may facilitatethe processing of the polyepitope in several epitopes or analogues andavoid the processing of overlapping epitopes.

The polyepitope of the invention enables the elicitation of a CTLresponse against at least one, especially against several epitopes oranalogues contained in the polyepitope, simultaneously, in a singleanimal or human.

In a particular embodiment, the polynucleotide encoding the polyepitopeof the invention further comprises a polynucleotide encoding a targetsignal, operably linked to the polynucleotidic unit encoding the mostN-terminal epitope of the at least two epitopes. “Operably linked” asused herein means that the target signal (upstream sequence) is linkedto the N-terminal epitope (downstream sequence) in a way enabling thetargeted signal to be operational, i.e., enabling to target thepolyepitope to the correct cellular compartment or domain. Therefore,the link between the two sequences allows each sequence to play its ownfunction in different locations and/or different stages. In a particularembodiment, said target signal is an endoplasmic reticulum signalsequence, and allows the polyepitope to be directed to the ER, forproper processing and association with the MHC class I molecule.

In a further embodiment, a codon encoding a methionine residue is addedupstream of the sequence encoding the most N-terminal epitope, to enablethe correct translation of the polynucleotide, if the translationalprocess requires an initiation codon. Such a codon is added, only whenthe most N-terminal epitope does not possess a methionine residue in itsfirst position.

The present invention also relates to a polynucleotide, according to thedefinitions given above, comprising or consisting of at least twopolynucleotide units selected from the group consisting of

SEQ ID NO: 21 a. ATGCCGCGCGCTCCCCGCTGCCGAGCC (n1), SEQ ID NO: 22b. GCTCCCCGCTGCCGAGCCGTGCGCTCCCTG (n4), SEQ ID NO: 23c. GCCCCCTCCTTCCGCCAGGTGTCCTGCCTG (n68), SEQ ID NO: 24d. AGACCCGCCGAAGAAGCCACCTCTTTG (n277), SEQ ID NO: 25e. CGGCCCTCCTTCCTACTCAGCTCTCTG (n342), SEQ ID NO: 26f. AGGCCCAGCCTGACTGGCGCTCGGAGGCTC (n351), SEQ ID NO: 27g. GACCCCCGTCGCCTGGTGCAGCTGCTC (n444), SEQ ID NO: 28h. TTCGTGCGGGCCTGCCTGCGCCGGCTG (n464), SEQ ID NO: 29i. GCTGGGAGGAACATGCGTCGCAAACTC (n966), SEQ ID NO: 30j. CTCCCGGGGACGACGCTGACTGCCCTG (n1107), SEQ ID NO: 31k. CTGCCCTCAGACTTCAAGACCATCCTG (n1123), and SEQ ID NO: 32l. GCCCCCCGTCGCCTGGTGCAGCTGCTC (n444*).

The following polynucleotides make also part of the invention:

-   -   a polynucleotide, comprising at least one polynucleotidic unit        selected from:

a. AGGCCCAGCCTGACTGGCGCTCGGAGGCTC (SEQ ID NO: 26) (n351)

b. GCCCCCTCCTTCCGCCAGGTGTCCTGCCTG (SEQ ID NO: 23) (n68),

c. GCTCCCCGCTGCCGAGCCGTGCGCTCCCTG (SEQ ID NO: 22) (n4),

d. GACCCCCGTCGCCTGGTGCAGCTGCTC (SEQ ID NO: 27) (n444),

e. TTCGTGCGGGCCTGCCTGCGCCGGCTG (SEQ ID NO: 28) (n464),

f. GCTGGGAGGAACATGCGTCGCAAACTC (SEQ ID NO: 29) (n966),

g. CTCCCGGGGACGACGCTGACTGCCCTG (SEQ ID NO: 30) (n1107),

h. CTGCCCTCAGACTTCAAGACCATCCTG (SEQ ID NO: 31) (n1123), and

i. GCCCCCCGTCGCCTGGTGCAGCTGCTC (SEQ ID NO: 32) (n444*), or theiranalogues as defined above, and at least one polynucleotidic unitselected from:

j. ATGCCGCGCGCTCCCCGCTGCCGAGCC (SEQ ID NO: 21) (n1),

k. AGACCCGCCGAAGAAGCCACCTCTTTG (SEQ ID NO: 24) (n277),

l. CGGCCCTCCTTCCTACTCAGCTCTCTG (SEQ ID NO: 25) (n342), or theiranalogues as defined above.

-   -   a polynucleotide, comprising at least one polynucleotidic unit        selected from:

a. AGGCCCAGCCTGACTGGCGCTCGGAGGCTC (SEQ ID NO: 26) (n351)

b. GCCCCCTCCTTCCGCCAGGTGTCCTGCCTG (SEQ ID NO: 23) (n68),

C. GCTCCCCGCTGCCGAGCCGTGCGCTCCCTG (SEQ ID NO: 22) (n4),

d. GACCCCCGTCGCCTGGTGCAGCTGCTC (SEQ ID NO: 27) (n444),

e. TTCGTGCGGGCCTGCCTGCGCCGGCTG (SEQ ID NO: 28) (n464),

f. GCTGGGAGGAACATGCGTCGCAAACTC (SEQ ID NO: 29) (n966),

g. CTCCCGGGGACGACGCTGACTGCCCTG (SEQ ID NO: 30) (n1107),

h. CTGCCCTCAGACTTCAAGACCATCCTG (SEQ ID NO: 31) (n1123), and

i. GCCCCCCGTCGCCTGGTGCAGCTGCTC (SEQ ID NO: 32) (n444*), or theiranalogues as defined above.

Are also included in the present invention, polynucleotides orpolynucleotide units encoding an epitope, analogue or polyepitope of theinvention, taking into consideration the degeneracy of the genetic code.Therefore, each amino acid can be encoded by the codon from the hTERTnucleotide reference sequence, or by any codon encoding said amino acid.

The invention also relates to a polynucleotide comprising or consistingof any combination of at least two of these polynucleotide units oranalogues thereof, selected from the above group, wherein saidpolynucleotide unit or analogue encodes a MHC class I-restricted,especially a HLA-B7-restricted, hTERT epitope.

All features given above concerning epitopes, analogues, polyepitopes,combination thereof, spacers, target signal sequences . . . areapplicable to any polypeptide of the invention as well as to thecorresponding polynucleotide sequences.

A recombinant vector, comprising or consisting of a polynucleotide ofthe invention, as defined above, is also one object of the presentinvention. The recombinant vector can be a vector for eucaryotic orprocaryotic expression, such as a plasmid, a phage for bacteriumintroduction, a YAC able to transform yeast, a viral vector andespecially a retroviral vector, or any expression vector. An expressionvector as defined herein is chosen to enable the production of anepitope or analogue or polyepitope as defined above, either in vitro orin vivo.

Therefore, besides the polynucleotide, the vector of the invention canfurther comprise transcription regulation regions (including promoter,enhancer, ribosome binding site (RBS), polyA signal), a terminationsignal, a prokaryotic or eukaryotic origin of replication and/or aselection gene. The features of the promoter can be easily determined bythe man skilled in the art in view of the expression needed, i.e.,constitutive, transitory or inducible, strong or weak, tissue-specificand/or developmental stage-specific promoter. Therefore, tissue-specificpromoters can be chosen depending on the organ in which a compositioncontaining this vector is administered, for example injected, anddepending on the intensity of expression required. In a particularembodiment, the promoter is the CMV promoter (human cytomegalovirus).Said vector can also comprise sequence enabling conditional expression,such as sequences of the Cre/Lox system or analogue systems.

The expression vectors of the invention may be viral vectors, andparticularly viral expression vector, such as retroviral-derived,especially lentiviral-derived vectors such as HIV-, FIV- or SIV-derivedvectors. More particularly, the lentiviral-derived vector is a humanlentiviral-derived vector such as an HIV expression vector, particularlyHIV-1 or HIV-2-derived vector. A retroviral-derived vector comprises aretroviral vector genome, usually included in a DNA construct, such as aplasmid, and expressed in viral particles, wherein said retroviralvector genome comprises the elements necessary for theretrotranscription, particularly the LTRs possibly mutated includingdeleted in part, especially deleted in the U3 region. In no case, theretroviral-derived vector contains all the nucleotide sequences encodingthe full-length retroviral proteins. Possibly, it contains part of oneor several of said nucleotide sequences providing it does not encodesaid proteins or functional fragments thereof. Said DNA constructcomprising said retroviral vector genome further comprises a DNA ofinterest recombined with the retroviral nucleotide sequences, said DNAof interest comprising or consisting of a polynucleotide of theinvention.

In a preferred embodiment, the retroviral-derived vector genomecomprises a DNA flap as described below and at least one polynucleotideof the invention. In a preferred embodiment, the retroviral-derivedvector is a HIV expression vector comprising a DNA flap as describedbelow and at least one polynucleotide of the invention. HIV vectorsexpress therefore only the nucleic acid(s) of interest, including thepolynucleotides of the invention, contained between the two HIV LTRs andcan thus accommodate large sequences up to 5-6 kb. A particularembodiment of the invention is a HIV expression vector, and mostparticularly a HIV-1 or HIV-2 expression vector, wherein a HIV-1 LTR orrespectively the HIV-2 LTR is deleted for the promoter and the enhancerof the U3 domain (ΔU3). This particular deletion has been previouslyshown to increase the expression of the nucleic acid(s) contained in thevector, and particularly when associated with a promoter. Anotherparticular embodiment, the vector comprises a LTR deleted in thepromoter and the enhancer of the U3 domain, a promoter such as a CMV orEF1α promoter and a polynucleotide of the invention.

In another embodiment, the polynucleotide of the invention introduced inthe retroviral-derived vector is included in an expression cassette.

A DNA flap (or Triplex as disclosed in WO 99/55892, WO 01/27300 and WO01/27304) is a nucleotide sequence of retroviral or retroviral-likeorigin comprising two essential regions, i.e., the cPPT (centralpolypurine tract) and the CTS (cis-acting termination region) regions,wherein the cPPT and CTS regions induce a three-stranded DNA structure.A DNA flap suitable for the invention may be obtained from a retrovirusor retrovirus-like organism such as retrotransposon, preparedsynthetically (chemical synthesis) or by amplification of the DNA flapfrom any retrovirus nucleic acid such as Polymerase Chain Reaction(PCR). The retrovirus, from which the DNA flap may be obtained, isparticularly a retrovirus or a lentivirus, especially a human retrovirusor lentivirus and is in particular a HIV retrovirus, the CAEV (CaprineArthritis Encephalitis Virus) virus, the EIAV (Equine Infectious AnaemiaVirus) virus, the VISNA virus, the SIV (Simian Immunodeficiency Virus)virus or the FIV (Feline Immunodeficiency Virus) virus. In a morepreferred embodiment, the DNA flap is obtained from an HIV retrovirus,for example HIV-1 or HIV-2 virus or any different isolate of these twotypes. It is noteworthy that the DNA flap is used isolated from itsnatural (viral genome) nucleotide context isolated from the pol gene inwhich it is naturally contained in a retrovirus. Therefore, the DNA flapis used, in the present invention, deleted from the unnecessary 5′ and3′ parts of the pol gene and is recombined with sequences of differentorigin.

The DNA flap acts as a cis-determinant of the vector nuclear import, andis of great interest for the recombination and the integration ofnucleic acid(s) into both non-dividing and dividing cells. Expressionretroviral-derived vector, especially HIV derived-vectors, including theDNA flap sequence (TRIP vectors) are able to transduce primary B and Tcells, macrophages, dendritic cells, etc with a tenfold higherefficiency than other HIV vectors that lack the DNA flap. A transductionof 80-90% of cells can be routinely obtained.

In a preferred embodiment, a vector suitable for an in vivo expressionand vaccine strategy is a retroviral and especially a lentiviral vector(see WO 99/55892, WO 01/27300 and WO 01/27304). Such vectors have beenshown to be particularly efficient and secure, when their genome ismodified (Firat et al. 2002, The Journal of Gene Medicine 4: 38-45).Indeed, these vectors have the ability to efficiently and stablytransfer therapeutic or reporter genes, in a large variety of cells andtissues, such as hematopoietic stem cells, brain, liver and retina.Moreover, this high transduction efficiency is irrespective of theproliferative status of the target cells. In particular, these vectorshave been shown to efficiently induce CD8⁺ T cell responses both in vivoin mice and ex vivo in humans, due to their capacity to transduceantigen presenting cells such as dendritic cell (DC) with highefficiency, ex vivo as well as in vivo (Esslinger et al. Hum Gene Ther2002; 13:1091-100; Breckpot et al. J Gene Med 2003; 5:654-67; Esslingeret al. J Clin Invest 2003; 111:1673-81).

The vector, defined in FIG. 7, has been used for in vitro or in vivoexpression of epitopes, analogues or polyepitopes of the invention. Asexamples of vectors, from which the vectors of the invention can bederived, are the following, all deposited with the CNCM (CollectionNationale de Culture de Microorganismes at Institut Pasteur, Paris,France):

Described in Accession Date of patent Vector name number depositionapplication pTRIP.EGFP I-2005 Apr. 15, 1998 WO PTRIP-MEL.IRES-GFP I-2185Apr. 20, 1999 99/55892 PTRIP-TEL/AML-IRES-GFP I-2326 Oct. 11, 1999 WOPTRIP-TEL/ILKE-IRES-GFP I-2327 Oct. 11, 1999 01/27300 PTRIP-DES-IRES-GFPI-2331 Oct. 11, 1999

The vectors described in the patent application WO 01/27304, andespecially TRIP ΔU3 Efα1 GFP and TRIP ΔU3 PL CMV GFP, can also be usedto derive the vectors of the present invention.

A particular vector of the invention is the pTRIP-CMV-ΔhTERT vector,deposited at the CNCM (Institut Pasteur, Paris, France) under the numberCNCM I-3660 on Jul. 28, 2006. A suitable growth medium for cultivatingthis vector is a TB medium, optionally supplemented with hygromycin.Another expression vector of the invention is the pTRIP-CMV-ΔhTERTvector deposited at the CNCM under the number CNCM I-3660 on Jul. 28,2006, in which the deleted hTERT sequence has been substituted by anypolynucleotide of the invention.

The present invention also relates to cells comprising thepolynucleotides or polynucleotide units of the invention.

In one embodiment, the cell is transfected with a vector of theinvention, by methods well known to the man skilled in the art, i.e. bychemical transfection (calcium phospate, lipofectamine), lipid-basedtechniques (liposome), electroporation, photoporation, use of viralvectors . . . . In another embodiment, a cell is transformed ortransduced with a polynucleotide of the invention, in a way enablingintegration of the polynucleotide in the cell genome either by arecombination with the homologous cellular sequence or by insertion inthe cellular genome. The transfection, infection or transduction canoccur ex vivo, i.e. in an artificial environment outside the livingorganism.

Among cells particularly interesting in the vaccine strategy are cellsof the immune system, and especially antigen presenting cells (APC). Ina particular embodiment, these cells are APCs involved either in MHCclass I recognition, like dendritic cells (DC) or in MHC class IIrecognition such as macrophages or lymphocytes B. Among DCs, ex vivofully maturated DCs, i.e., DCs that have been in vitro maturated byepitopes or analogues, are preferred.

As used herein, the terms “transfected”, “transformed” or infected”refer to a cell comprising a vector of the invention (transientexpression), whereas the term “genetically transformed” refers to a cellwhose genome has been definitively modified by a polynucleotide of theinvention (permanent expression).

Said transitory or stably transformed cells can be any prokaryotic(bacteria) or eukaryotic (yeast, animal including mammal especiallyhuman) cells. In an embodiment, cells are non-human cells. In aparticular embodiment, cells of the invention are isolated human cells,“isolated” meaning outside of its natural environment.

A particular host is the E. coli strain deposited at the CNCM under thenumber CNCM I-3660 on Jul. 28, 2006.

The invention also relates to epitopes, analogues or polyepitope asdefined above when describing the polynucleotides of the invention andparticularly to any polypeptide encoded by a polynucleotide orpolynucleotide units of the invention. Particular polypeptides are MHCclass I-restricted, especially HLA-87-restricted, hTERT epitope, chosenfrom the group consisting of:

SEQ ID NO: 6 a. MPRAPRCRA (p1), SEQ ID NO: 7 b. APRCRAVRSL (p4),SEQ ID NO: 8 c. APSFRQVSCL (p68), SEQ ID NO: 9 d. RPAEEATSL (p277),SEQ ID NO: 10 e. RPSFLLSSL (p342), SEQ ID NO: 11 f. RPSLTGARRL (p351),SEQ ID NO: 12 g. DPRRLVQLL (p444), SEQ ID NO: 13 h. FVRACLRRL (p464),SEQ ID NO: 14 i. AGRNMRRKL (p966), SEQ ID NO: 15 j. LPGTTLTAL (p1107),and SEQ ID NO: 16 k. LPSPKFTIL (p1123).

Particular HLA-87-restricted hTERT epitopes, are chosen from the groupconsisting of:

SEQ ID NO: 11 a. RPSLTGARRL (p351), SEQ ID NO: 8 b. APSFRQVSCL (p68),SEQ ID NO: 7 c. APRCRAVRSL (p4), SEQ ID NO: 12 d. DPRRLVQLL (p444),SEQ ID NO: 13 e. FVRACLRRL (p464), SEQ ID NO: 14 f. AGRNMRRKL (p966),SEQ ID NO: 15 g. LPGTTLTAL (p1107), SEQ ID NO: 16 h. LPSPKFTIL (p1123),and SEQ ID NO: 17 i. APRRLVQLL (p444*).

A particular group consists of the following MHC class I-restricted,especially HLA-87-restricted, hTERT epitopes:

SEQ ID NO: 6 a. MPRAPRCRA (p1), SEQ ID NO: 7 b. APRCRAVRSL (p4),SEQ ID NO: 8 c. APSFRQVSCL (p68), and SEQ ID NO: 11d. RPSLTGARRL (p351).

Another group consists of the following HLA-87-restricted hTERTepitopes: RPAEEATSL (SEQ ID NO: 9) (p277) and RPSFLLSSL (SEQ ID NO: 10)(p342). A preferred HLA-87 allele targeted by these epitopes isHLA-80702.

The invention also concerns analogues of the peptides disclosed above,and having at least one amino acid substitution. Features pertaining tothese analogues have especially been described in the above pages. Aparticular peptide analogue is p444* having the following peptidesequence APRRLVQLL (SEQ ID NO: 17).

Finally, the invention also concerns any polynucleotide encoding a hTERTepitope, analogue or polyepitope as described in the presentspecification.

The invention also relates to a polyepitope comprising at least twoepitopes and/or analogues as described above. The polyepitopes of theinvention are not the full-length hTERT protein. The size of saidpolyepitope can range from 15 to 1000 in particular from 50 or from 100to 1000 amino acids, especially and particularly about 100, 200, 300,400, 500 or 1000 amino acids. Such an epitope comprises or consists of 2to 30 epitopes or analogues, and particularly 2 to 20 or 2 to 10epitopes and/or analogues. A particular polyepitope comprises orconsists of 6 consecutive epitopes and has the following sequence:MPRAPRCRAAPRCRAVRSLAPSFRQVSCLRPAEEATSLRPSFLLSSLRPSLTGARRL (SEQ ID NO:20). Another particular polyepitope comprises or consists of the p1, p4,p68, p277, p342 and p351 epitopes, the epitopes being either consecutiveto each other in the polyepitope obtained or all or part of them beingseparated by peptide spacers.

Another polyepitope of the invention comprises at least two epitopes, atleast one being chosen from the group consisting of:

SEQ ID NO: 11 a. RPSLTGARRL (p351), SEQ ID NO: 8 b. APSFRQVSCL (p68),SEQ ID NO: 7 c. APRCRAVRSL (p4), SEQ ID NO: 12 d. DPRRLVQLL (p444),SEQ ID NO: 13 e. FVRACLRRL (p464), SEQ ID NO: 14 f. AGRNMRRKL (p966),SEQ ID NO: 15 g. LPGTTLTAL (p1107), SEQ ID NO: 16 h. LPSPKFTIL (p1123),and SEQ ID NO: 17 i. APRRLVQLL (p444*),

or analogues thereof obtained by substitution of at least one amino acidresidue, and at least one being chosen from the group consisting of

SEQ ID NO: 6 j. MPRAPRCRA (p1), SEQ ID NO: 9 k. RPAEEATSL (p277),SEQ ID NO: 10 l. RPSFLLSSL (p342),or analogues thereof obtained by substitution of at least one amino acidresidue, wherein said polyepitope is not the full length hTERT.

Polypeptides of the invention can be synthesized chemically, or producedeither in vitro (cell free system) or in vivo after expression of thecorresponding nucleic acid sequence in a cell system.

The full-length hTERT protein (SEQ ID NO: 37) as represented in FIG. 1is excluded from the invention, as well as the corresponding full-lengthcoding sequence (SEQ ID NO: 36). Also excluded from the presentinvention, the RPALL TSRL (SEQ ID NO: 42) peptide.

These peptides are excluded particularly in the context of HLA-87recognition.

The invention also concerns an epitope, analogue, polyepitope orpolynucleotide, an expression vector or host cell as defined above, foruse to elicit or participate in providing a HLA-B7-restricted immuneresponse against hTERT.

The invention also concerns a composition comprising a polynucleotide, avector, a host cell and/or a polypeptide of the invention. In aparticular embodiment, said composition is suitable for in vivoadministration, i.e., said composition is prepared for injection, ormore generally for association with physiologically-acceptable liquidsolutions or emulsions for administration. Said composition may be usedeither for systemic or local administration, especially by injection,and may further comprises a pharmaceutically suitable excipient(including water, saline, dextrose, glycerol, ethanol, and combinationsthereof) or a carrier and/or a vehicle.

In a particular embodiment, said composition comprises a polynucleotideof the invention encoding an epitope, analogue or encoding a polyepitopeas described above. Said composition can comprise other nucleic acidmolecules encoding at least one hTERT epitope or analogue thereof orpolyepitope, restricted to a different MHC class I allele from that ofHLA-B7. The combination of hTERT epitopes restricted to different HLAsupertypes or alleles enables covering a larger population of patientsin need of treatment than a sole supertype or allele. To this end,HLA-A1, -A2, -A3 and -A24 are preferred.

In another embodiment, the composition comprises nucleic acid moleculesencoding at least one hTERT epitope or analogue thereof or polyepitope,restricted to MHC class II. Said composition can comprise anycombination of nucleic acid molecules as described above, with at leastone HLA-B7-restricted hTERT epitope, analogue or polyepitope of thepresent invention. The combination, in a composition of nucleic acidmolecules, of polynucleotides encoding Class I and Class II-restrictedepitopes enabling the reaction of various immune cells (T lymphocytes orNK cells for class I versus helper lymphocytes for class II), and/or theelicitation of various immune responses (humoral versus cellularresponse) is also within the present invention.

In another embodiment, the composition comprises nucleic acid moleculescomprising at least such molecules encoding one tumour-specific antigen(TSA) and/or at least one tumour-associated antigen (TAA), such asprostate specific antigen (PSA), prostate-specific membrane antigen(PSMA) or prostatic acid phosphatase (PAP) (Tartour et al. 2000 ImmunolLett September 15; 74(1): 1-3; Tartour et al. 1996 Presse Med. November16; 25(25): 1717-22).

Several types of therapeutic compositions can be used to elicit animmune response against an epitope or analogue of the invention.

A composition comprising a polynucleotide of the invention isadministered to a host, for instance injected (known as DNA vaccination)and said nucleic acid expresses in vivo a polypeptide comprising orconsisting of multiple epitopes according to the invention. Such DNAvaccines usually consist of plasmid vectors as described above. Thedelivery of naked DNA has shown to be poorly efficient, and somecarriers are needed to improve the delivery and uptake of DNA intocells. Two types of carriers have been yet developed: (1) viral carriers(adenoviruses, lentiviruses, measles virus), or (2) non-viral carrierssuch as polymers (and especially cationic polymers), encapsulated-DNA(liposomes, comprising cationic lipids interact spontaneously andrapidly with polyanions, such as DNA and RNA, resulting inliposome/nucleic acid complexes) or DNA linked to gold microparticles.Moreover, agents, which assist in the cellular uptake of nucleic acid,such as calcium ions, bacterial proteins (Shigella proteins) viralproteins and other transfection facilitating agents, may advantageouslybe used. Another type of composition according to the invention is acomposition comprising lentiviral pseudoparticles comprising a vector orvector genome as mentioned above.

Another type of composition comprises an epitope, analogue orpolyepitope of the invention. Such a composition is immunogenic, i.e.,it is capable of eliciting an immune response in a host in which it isadministered. However, to increase the immunogenic properties of thepolypeptides of the invention, an adjuvant can be administered with thepolypeptide, to elicit or improve the immune response. An adjuvant isdefined as any substance that enhances the immunogenicity of an antigenmixed with said adjuvant. Some adjuvants convert soluble antigens intosmall particles, such as aluminium hydroxide gel, oil in water emulsionor immune stimulatory complexes (ISCOMs). Another class of adjuvantscomprises sterile constituents of bacteria such as cell wall orpolysaccharides, Freund adjuvant. Finally, emulsifying agents or pHbuffering agents can also be used to enhance the immunogenic behaviourof the epitope or analogue.

All compositions quoted above can be injected in a host via differentroutes: subcutaneous (s.c.), intradermal (i.d.), intramuscular (i.m.) orintravenous (i.v.) injection, oral administration and mucosaladministration, especially intranasal administration or inhalation. Thequantity to be administered (dosage) depends on the subject to betreated, including the condition of the patient, the state of theindividual's immune system, the route of administration and the size ofthe host. Suitable dosages range from 200 μg to 1 mg, and can bemodified by one skilled in the art, depending on circumstances.

The compositions of the invention are useful for the prophylaxis and ortreatment of malignant states in patients, resulting from uncontrolledcell proliferation, including tumors, resulting from the over-expressionof hTERT, as well for the treatment of detrimental consequencesaccompanying such malignant state, e.g., cancer. The expression“treatment” encompasses the curative effect achieved with compositionsof the invention and also the beneficial effect for the patientundergoing the treatment, said effect being either obtained at cellularlevel or clinical level, including as a result, an improvement of thecondition of the patient and/or a remission state or a recovery of ahealth state. In a particular embodiment, the composition of theinvention further comprises additional active compounds useful for theprophylaxis or the treatment of tumors, either general compounds orcompounds proved to be active in a tissue-specific cancer.

The invention also concerns a process to activate T lymphocytes againstclass I-restricted, particularly HLA-B7-restricted, hTERT epitopes:

-   -   a. providing T lymphocytes, and,    -   b. in vitro cultivating said T lymphocytes with at least one        epitope or epitope analogue or polyepitope of the invention, in        conditions enabling the activation of said lymphocytes.        In a particular embodiment, activated T lymphocytes are        cytotoxic T lymphocytes (CTL). Conventional conditions to        activate T lymphocytes use interleukine (IL) 2, IL-7, IL-12        and/or IL-15, and are described in Minev et al. (2000 PNAS;        97(9): 4796-4801) incorporated herein by reference.

The invention also relates to a process to check the immunogenicbehaviour of a hTERT peptide, comprising:

-   -   a. activating T lymphocytes, by in vitro cultivating said T        lymphocytes with at least one epitope or epitope analogue or        polyepitope of the invention, in appropriate conditions,    -   b. in vitro cultivating said activated lymphocytes with target        cells expressing, at their cell surface, a hTERT epitope of the        invention bound to a MHC-class I molecule, in suitable        conditions, and    -   c. determining whether said activated lymphocytes react against        said target cells.

In a particular process to check the immunogenic behaviour of a hTERTpeptide, the epitope when used individually i.e., not under the form ofa polyepitope, is chosen among (a) RPSLTGARRL (SEQ ID NO: 11) (p351),(b) APSFRQVSCL (SEQ ID NO: 8) (p68), (c) APRCRAVRSL (SEQ ID NO: 7) (p4),(d) DPRRLVQLL (SEQ ID NO: 12) (p444), (e) FVRACLRRL (SEQ ID NO: 13)(p464), (f) AGRNMRRKL (SEQ ID NO: 14) (p966), (g) LPGTTL TAL (SEQ ID NO:15) (p1107) and (h) LPSPKFTIL (SEQ ID NO: 16) (p1123); an example ofanalogue is the p444* as defined above.

A particular vector of the invention is the pTRIP-CMV-ΔhTERT vector,deposited at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES(CNCM) (Institut Pasteur, 25 rue du Docteur Roux F-75724 Paris, France)under the number CNCM 1-3660 on Jul. 28, 2006. A suitable growth mediumfor cultivating this vector is a TB medium, optionally supplemented withhygromycin. Another expression vector of the invention is thepTRIP-CMV-ΔhTERT vector deposited at the CNCM under the number CNCM1-3660 on Jul. 28, 2006, in which the deleted hTERT sequence has beensubstituted by any polynucleotide of the invention.

The activation of lymphocytes includes the presentation of said epitopesor analogues by antigen presenting cells to naïve (not activated) Tlymphocytes. Once naïve T lymphocytes have recognized the epitope oranalogue of the invention, in the context of a particular class-I HLAmolecule, they are said “activated” and ready to recognize said epitopeon the cell surface of a target cell. The contact between said activatedlymphocytes (effector cells) and target cells (expressing the epitope,from which the lymphocyte has been activated), leads to the secretion ofmolecules and killing of the target cells. If an epitope or analogueused to activate lymphocytes leads to an efficient destruction of atarget cell bearing said epitope by said activated lymphocytes, saidepitope can be considered as immunogenic enough to allow not only invitro but also in vivo T cell reaction against cells expressing saidepitopes. Suitable conditions for target cells/lymphocytes recognitionare a 4 hour-contact at 37° C. in RPMI medium.

The invention also relates to a process to in vitro maturate cells, andespecially antigen presenting cells (APC), B cells, T cells and/ordendritic cells, against MHC class I-restricted, particularlyHLA-B7-restricted, hTERT epitopes. Such a process to in vitro maturatecells comprises:

-   -   a. providing cells,    -   b. enabling the maturation of said cells with at least one MHC        class I-restricted, particularly HLA-B7-restricted, hTERT        epitope or epitope analogue or polyepitope of the invention, and    -   c. optionally, favouring the expansion of said maturated cells.

As described above, the activation of lymphocytes requires epitopepresentation by maturated antigen presenting cells. In a preferredembodiment of the invention, said cells express at least one HLA-B7allele. One of them, dendritic cells (DC), are particularly efficient inpresentation of endogenous epitopes restricted to MHC class I, to Tlymphocytes. One of the objectives in the maturation of said cells istheir administration once maturated, to a patient in need of treatment.The administration of said maturated DCs would result in vivo in theactivation of patient's lymphocytes, and rapid reaction against cellexpressing the epitope (the one, the DCs have been transformed with).

In a particular embodiment, a process to in vitro maturate dendriticcells comprises:

-   -   a. providing dendritic cells,    -   b. enabling the maturation of said dendritic cells with at least        one MHC class I-restricted hTERT epitope or epitope analogue or        polyepitope of the invention, and    -   c. optionally, favouring the expansion of said maturated        dendritic cells.

In a particular embodiment, said dendritic cells are isolated fromeither circulating blood or bone marrow cells. In another embodiment,dendritic cells are isolated from the patient in need of treatment orfrom an HLA-matched donor, to avoid rejection after the administrationto said patient.

The maturation of DCs can be achieved by genetic transformation of saiddendritic cells with a polynucleotide of the invention, by transfectionof said dendritic cells with a vector of the invention or by contactingsaid dendritic cells with at least one epitope, epitope analogue orpolyepitope of the invention. The genetic transformation is preferredbecause of its efficiency and the permanent expression of the epitope,analogue or polyepitope encoded by the polynucleotide inserted in the DCgenome.

The invention also concerns a polynucleotide encoding aHLA-87-restricted hTERT epitope for use in the prevention and/ortreatment of cancer. In a particular embodiment, said polynucleotide,for use in the prevention and/or treatment of cancer encodes aHLA-87-restricted hTERT epitope or analogue thereof or a polyepitopecomprising at least one HLA-87-restricted hTERT epitope or analoguethereof as described in the present application. As far as thepolyepitope-encoding polynucleotide is concerned, it does not coincidewith the coding sequence of the full-length hTERT. On the other hand, itcan coincide with a mutated or deleted version of HTERT, said mutationor deletion suppressing the catalytic activity of the human telomerase.In a particular embodiment, said polynucleotide comprises at least onepolynucleotide unit encoding a HLA-87-restricted hTERT epitope, chosenfrom the group consisting of:

SEQ ID NO: 6 a. MPRAPRCRA  (p1), SEQ ID NO: 7 b. APRCRAVRSL (p4),SEQ ID NO: 8 c. APSFRQVSCL (p68), SEQ ID NO: 9 d. RPAEEATSL  (p277),SEQ ID NO: 10 e. RPSFLLSSL  (p342), SEQ ID NO: 11 f. RPSLTGARRL (p351)SEQ ID NO: 12 g. DPRRLVQLL  (p444), SEQ ID NO: 13 h. FVRACLRRL  (p464),SEQ ID NO: 14 i. AGRNMRRKL  (p966), SEQ ID NO: 15 j. LPGTTLTAL  (p1107),and SEQ ID NO: 16 k. LPSPKFTIL  (p1123).or at least one analogue of said HLA-87-restricted hTERT, such asAPRRLVQLL (SEQ ID NO: 17) (p444*), or any combination forming apolynucleotide having at least two polynucleotide units encoding saidHLA-87 epitopes and/or analogues.

The invention concerns a HLA-B7 hTERT epitope, and especially aHLA-B0702 restricted epitope for use in the prevention and/or treatmentof cancer. The invention relates also to a polynucleotide, a vector, ahost cell or a polypeptide of the invention for use in the preventionand/or treatment of cancer.

The invention also relates to the use of a HLA-B7 hTERT epitope, (orcorresponding polynucleotide) for the manufacture of a drug for theprevention and/or treatment of cancer. Particular HLA-B7 hTERT epitopes(or corresponding polynucleotides) are those described above as well asvectors, cells or compositions comprising or consisting of them. In aparticular embodiment, the use of polynucleotide, vector, host cell orpolypeptide comprising or consisting of a polyepitope of the inventionin the manufacture of a drug for the prevention and/or treatment ofcancer is intended for patients having at least one HLA-B7 allele asdefined above, and particularly at least one HLA-B0702 allele.

Each definition provided in the specification applies to each and anypeptide (epitope, analogue or polyepitope) as well as to each and anypolynucleotide, taken individually (as such) or encompassed in a group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Gene encoding the hTERT protein (SEQ ID NO: 36) andcorresponding amino acid sequence (SEQ ID NO: 37).

The coding sequence is located between the nucleotide 56 and 3454.Initiation and termination codons are underlined. First line is thenucleotide sequence; second line is the corresponding amino acidsequence. Third line is the numerotation of the hTERT coding sequence,starting from the initiation codon as the first amino acid.

FIG. 2: hTERT derived peptides are processed in HLA-B0702 transgenicmice.

HLA-B7 Tg mice and one naïve mice (N) were immunized with 100 μg DNAencoding Htert. On day 14, spleen cells from each mouse were separatelyin vitro stimulated with different hTERT-derived peptides. Effectorcells were essayed 6 days later against RMA-B7 targets loaded withrelevant (▪) or control (□) peptides as described in the material andmethods. Percentage of lysis at a 60/1 ratio are shown (results from twoindependent experiments).

FIG. 3: Induction of CTL response against hTERT in PBMC from healthblood donors.

T-lymphocyte cells from HLA-*B0702⁺ healthy donors were activated witheach of the six hTERT peptide-pulsed autologous PBMC as detailed inmaterials and Method. After four rounds of weekly stimulation, effectorcells, pulsed with relevant (▪) or control (□) peptides, were essayedfor lytic activity against ⁵¹Cr-labeled T2-B7 cells. Percentage of lysisat a 20/1 effector-target ratio is shown. Results from 8 out of 10donors are presented (d1 to d8).

FIG. 4: Effect of an anti-HLA class I mAb on cytotoxicity of CTLp351against tumor cells.

The cytotoxicity of the CTLp351 line against HLA-*B0702⁺ tumor celllines Mamo and U293T pre-treated either in absence (none) or presence ofHLA mAbs (anti-HLA class I mAb or an anti-class II mAb (HLA-DR)) wasdetermined by standard ⁵¹Cr-release assay at effector-target ratio of10/1.

FIG. 5: Ex-vivo detection of hTERT-specific T-cell response afterLv-hTERT immunization.

A) HLA-B7 transgenic mice were immunized with recombinant Trip-hTERTparticles or control Trip-GFP (1500 ng). After 12 days, hTERTpeptide-specific T cells producing IFNγ of each mouse were detected exvivo by IFNγ-ELISPOT assay within freshly spleen cells. The number ofIFNγ SFCs was calculated after subtracting negative control values.Results from three independent experiments are represented.

B) HHD mice were immunized with Trip-hTERT as described above. hTERTpeptide-specific T cells producing IFNγ were detected ex vivo by ELISPOTas described above. Results from two independent experiments arerepresented.

FIG. 6: Priming of specific CD8⁺ T cells responses in HLA-B*0702transgenic mice following Trip-hTERT immunization

HLA-B*0702 transgenic mice were immunized either with Trip-hTERT (4first black bars) or control (2 last horizontal vertical stripe bars).12 days later, IFN-γ producing-single cells within splenocytes of eachmouse were detected ex vivo by IFN-γ ELISPOT assay. FICOLL® purifiedlymphocytes from freshly isolated splenocytes of individual immunizedmice were directly cultured with or without 5 μg/ml of eachHLA-B*0702-restricted hTERT-derived peptides for 24 h. The number ofspecific IFN-γ SFCs was calculated after subtracting non-specific valuesobtained with control without peptide (<15 SFC), and the responses wereconsidered positive for SFC≧30.

FIG. 7: Schematic representation of the pTRIP-hTERT

This lentiviral-derived vector contains the psi sequence, the cPPT andCTS central cis-active sequences (Flap) of the HIV-1 genome and the CMVpromoter which allows the expression of the gene of interest. Moreover,the U3 domain is deleted in the 3′LTR (ΔU3).

FIG. 8:

A) DNA pTRIP-CMV-ΔhTERT immunization primed hTERT-specific CD8⁺ T cellsresponses in HHD mice. HHD mice (HLA-A2.1 Tg) were DNA immunized with aDNA encoding a non-functional form of HTERT (pTRIP-CMV-ΔhTERT). Ten dayslater, IFN-γ producing peptide-specific T cells were detected ex vivo byIFN-γ-ELISPOT assay. FICOLL® purified lymphocytes from splenocytes ofindividual immunized mouse were directly cultured for 24 h, with orwithout 5 μg/ml of each HLA-A2.1-restricted hTERT-derived peptide. Thenumber of specific-IFN-γ SFCs was calculated as described above.Responses were considered positive for SFC≧30.

B) Induction of short CTL responses in HHD mice after pTRIP-CMV-ΔhTERTimmunization. HHD mice were immunized with a DNA encoding anon-functional form of HTERT (pTRIP-CMV-ΔhTERT) for 10 days. Spleencells from individual mice were restimulated in vitro withHLA-A2.1-restricted hTERT-derived p540 and pY572 peptides for 6 days.Effector cells were tested in a ⁵¹Cr-release assay againstHHD-transfected EL4 cells loaded with either the relevant peptide or theirrelevant peptide.

FIG. 9: Sequence of a non-functional hTERT protein (deletion of aminoacids 867 to 869): DNA sequence disclosed as SEQ ID NO: 38 and aminoacid sequence disclosed as SEQ ID NO: 39.

The coding sequence is located between the nucleotide 59 and 3348.Initiation and termination codons are underlined. First line is thenucleotide sequence; second line is the corresponding amino acidsequence. Third line is the numerotation of the hTERT coding sequence,starting from the initiation codon as the first amino acid.

FIG. 10: Sequence of a non-functional hTERT protein (deletion of aminoacids 864 to 872). DNA sequence disclosed as SEQ ID NO: 40 and aminoacid sequence disclosed as SEQ ID NO: 41.

The coding sequence is located between the nucleotide 59 and 3438.Initiation and termination codons are underlined. First line is thenucleotide sequence; second line is the corresponding amino acidsequence. Third line is the numerotation of the hTERT coding sequence,starting from the initiation codon as the first amino acid.

EXAMPLES I—Materials and Methods

Blood Donors

Peripheral bloods samples were obtained following written informedconsent from adult healthy platelet donors (centre de transfusionsanguine de l'hôpital Mondor, Créteil, France). HLA typing of peripheralblood donors was performed in the HLA laboratory of the H. Mondor.Hospital Creteil (France). The study was approved by the French BloodBank Institute.

Mice

HLA-*B0702 transgenic (Tg) mice, expressing an HLA-B0702 α1α2, H2-Kd α3chimeric construct, in combination with constitutive murine β2-mmolecule (HLA-B7^(mα3)) and HHD transgenic mice expressing a chimericHLA-A2.1/H2-Db molecule, were deleted of their H2-Db and H2-k^(b) genesas previously described (Pascolo et al. J Exp Med 1997; 185:2043-51;Rohrlich et al. Int Immunol 2003; 15:765-72). These mice are on aC57BL/6 background and were bred and maintained under specificpathogen-free conditions in our animal facility.

Tumor Cells Lines

The T-B hybrid. T1, EBV-transformed B cell JY, renal cancer cell lineU293T and Burkitt lymphoma cell Raji were from American type CultureCollection (ATCC). Melanoma cell lines (SK23MEL, LB34, and KUL68) werekindly provided by P. Coulie (Bruxell, Belgium) and EBV-transformed Bcell BBG.1 and BC3 were kindly provided by H. Collandre (R.A.H.P.,Grenoble, France).

HLA-*B0702 transfected TAP deficient T2 cells (T2-B7) were kindlyprovided by P. Cresswell (Smith et al. J Immunol 1996; 156:3755-64).Murine lymphoma cell lines RMA, and EL4 were from ATCC; theses cellswere also transfected with HLA-*B0702 gene and used as target cells

Epitope Selection Peptide Synthesis

We used predictive algorithm “SYFPEITHI” (Lu and Celis E Cancer Res2000; 60:5223-7) to analyse amino acid sequence of hTERT for theexistence of 9-amino acid (nonamer) or 10-amino acid (decamer) peptides,predicted to bind to HLA-*80702. We selected candidate peptides thatcontain canonical HLA-87-binding anchors, Pro at position 2 andhydrophobic aliphatic (Ala or Leu) at carboxyl-termini, and according totheir highest predictive score. Six peptides were retained andsynthesized, three 9-amino acid peptides named p1, (MPRAPRCRA (SEQ IDNO: 6), residues 1-9), p277 (RPAEEATSL (SEQ ID NO: 9), residues 277-285)and p342 (RPSFLLSSL (SEQ ID NO: 10), residues 342-350), and three10-amino acid peptides, p4 (APRCRAVRSL (SEQ ID NO: 7), residues 4-13)p68, (APSFRQVSCL SEQ ID NO: 8, residues 68-77), and p351 (RPSLTGARRL(SEQ ID NO: 11), residues 351-360) (anchor positions are underlined).

Peptides derived from human cytomegalovirus pp65, RPHERNGFYV (SEQ ID NO:33) (R1OTV), and human immunodeficiency virus type 1 IPRRIRQGL (SEQ IDNO: 34) were synthesized and were used as control peptides. Peptidederived from hepatitis B virus core 128-140 (TPPATRPPNAPIL (SEQ ID NO:35)) was used as helper peptide for peptide immunization in mice.Peptides were purchased from PRIM to a minimum purity 80% and werereconstituted in distilled water or DMSO at a concentration of 2 mg/ml.

HLA-B0702 Binding/Stabilization Assay

The relative avidity of hTERT derived peptides for HLA-*B0702 wasmeasured by using a MHC stabilization assay on HLAB0702 transfected T2(T2-B7) cells in comparison with a reference peptide (R10TV) asdescribed (Rohrlich et al. Int Immunol 2003; 15:765-72). Briefly, T2-B7were incubated overnight at 2×10⁵ cells/well in 96-well plates in serumfree medium AIM-V (Invitrogen Corp., Gibco), supplemented with 100 ng/mlof human β2-microglobulin, in the absence (negative control) or in thepresence of either reference peptide R10V or hTERT peptides at variousfinal concentrations (100, 10, 1 and 0.1 μM). T2-B7 cells were labelledwith a saturating concentration of ME.1 an anti-HLA-B7 mAb, then washedtwice and finally stained with FITC-conjugated F(ab′)₂ goat anti-mouseIg before flow cytometry.

Results are expressed as values of relative avidity, that is the ratioof concentration of test peptide necessary to reach 20% of the maximalbinding (obtained with the reference peptide) over the concentration ofthe reference peptide. Therefore, the lower the value, the stronger thebinding.

Peptide Immunization of HLA-*B0702 Transgenic Mice for CTL Induction

Female HLA-*B0702 transgenic mice at 8-10 weeks of age were injectedsubcutaneously (s.c.) at the base of the tail with 50 μg of individualHLA-B0702 restricted hTERT peptides supplemented with 140 μg of helperpeptide co-emulsified in incomplete Freund's adjuvant (Difco, Detroit,Mich.). Ten days later, spleen cells of individual mouse werere-activated in vitro with relevant peptide in six wells plate. EffectorCTL cells were tested in a standard 4-5 h ⁵¹Cr-release assay, usingrelevant or negative control peptide-pulsed, HLA-*B0702 transfected RMAcells (RMA-B7). Mice were considered as responders, when specificlysis≧10% was observed.

DNA Immunization in HLA-*B0702 Transgenic Mice

The LvCMV-hTERT plasmid vector encoding the hTERT gene under the controlof CMV promotor was purified on plasmid GIGA kit columns underendotoxin-free conditions (QIAGEN®). Anesthetized HLA-*B0702 Transgenicmice were injected with said plasmid (50 μg each side) into regeneratingtibialis anterior muscles. 14 days after, spleen cells of individualmouse were re-activated in vitro with peptide-pulsed (10 μg/ml),syngenic γ-irradiated (50 Gy) LPS-lymphoblast in complete medium,supplemented with 10% supernatant from Con A-activated rat spleenscells. Cytotoxicity assays were performed 6 days as described.

Lentiviral Vector Construct and Production

The pTRIP-deltaU3-CMV-hTERT (referred as TRIPLv-hTERT or Lv-hTERT orpTrip-hTERT) (FIG. 7) construct was created by first subcloning anEcoRI-SaII hTERT insert derived from the pBABE-hygro-hTERT plasmid(Counter et al. Proc Natl Acad Sci U.S.A. 1998; 95:14723-8) into thepSP73 vector (Promega). A BgIII-SaII fragment was then inserted into thepTRIP-CMV plasmid cut with BamH1 and XhoI. Pseudo typed recombinantretroviral particles were produced by transient (48 h) transfection of293 T cells as described (Zennou et al. Cell 2000; 14; 101:173; Firat etal. J Gene Med 2002; 4:38-45). The recombinant retroviral particles wereconcentrated by ultra-centrifugation and resuspended in PBS. The amountof vector particles was estimated from that of p24 protein in acommercially available ELISA assay (NEN, DUPONT, France Perkin Elmer).

The pTRIP-CMV-ΔhTERT vector, deposited at the CNCM (Institut Pasteur,Paris, France) under the number CNCM 1-3660 on Jul. 28, 2006, wascarried out as described in the paragraph above. However, the hTERTprotein was rendered non-functional by deletion of amino acids 867 to869, corresponding to nucleotides 2654 to 2662 of FIG. 1 (wild-type).The catalytically dead hTERT RT mutant (ΔhTERT) was generated bycreating a deletion of amino acid residues 867 to 869 using theQUICKCHANGE® XL Site-Directed Mutagenesis Kit (STRATAGENE®) and verifiedby sequencing.

Immunization in MHC Class I Transgenic Mice and CTL Detection

Immunization with TRIPLv-hTERT was performed as a single subcutaneously(at the base of the tail) injection of 1500 ng of TripLv-hTERTsuspension or control vector.

Immunization was performed in HLA.A2 transgenic mice as a singleintraperitoneal injection of recombinant lentiviral particles,pTRIP-CMV-ΔhTERT or Trip-GFP as a control, equivalent to 1500 ng of p24antigen in 500 μl of PBS.

12 days later, hTERT peptide-specific T among splenocytes were detectedby an ELISPOT assay (see below). Cytotoxicity assays were performed onthe same immune splenocyte populations after in vitro stimulation withpeptide-pulsed as described above.

Evaluation of T-Cell Response by Ex Vivo IFN-γ ELISPOT Assay

Peptide-specific T cells from immunized mice were detected by IFN-γELISPOT assay as previously described (Miyahira et al. J Immunol Methods1995; 181:45-54). Anti-mouse IFN-γ mAb's (3 μg/ml; Pharmigen, BectonDickinson biosciences) were coated onto 96-well nitrocellulosemicroplates (multi screen; Millipore corp, Molsheim, France). After redcell lysis, freshly isolate spleen lymphocytes of individual mouse(5×10⁵, 2.5×10⁵ and 1.25×10⁵ cells/well) were directly cultured with orwithout 5 μg of native hTERT peptide for 18 h at 37° C. After washings,the plates were incubated 2 hours with biotinylated anti-mouse IFN-γ (2μg/ml; Pharmigen, Becton Dickinson biosciences). Finally, the plateswere washed and incubated at 37° C. for 1 h with alkalinephosphatase-conjugate streptavidin (Roche molecular biochemicals,Mannheim, germany). Positive controls include cells stimulated withphorbol myristate acetate (100 ng/ml, Sigma)) and ionomycin (1 μg/ml).IFNγ spot-forming cells (SFCs) were developed by adding peroxidasesubstrates (BCIP/NBT, Promega Corp, Madison W; USA) and counted usingautomated image analysis system a Bioreader 2000 (Biosys, Karben,germany). The number of specific SFCs was calculated after subtractingnegative control values (<10 SFC). Responses were positive if the meanof SFCs in stimulated well was greater than the mean+2 S.D. of the SFCsin the negative control wells and greater than 50 SFC/10⁶ cells.

Cytolytic Assay

Cytotoxicity assays were performed by using standard 4-5 h ⁵¹Cr releaseassay as previously described (Firat et al. J Gene Med 2002; 4:38-45).Specific lysis in % was calculated by subtracting non-specific lysisobserved with the control peptide. Mice were considered as responserswhen specific lysis 10% was observed.

Generation of hTERT Peptide-Specific CTL in Human

Human CTL from donors were obtained after in vitro reactivated PBMC for4 weeks with hTERT peptide HLA-B0702 restricted as described previously(Hernandez et al. Proc Natl Acad Sci U.S.A. 2002; 99:12275-80). Briefly,Ficoll FICOLL®-purified human PBMCS were thawed and incubated(4×10⁶/well) in 24-well plates in RPMI 1640, 1 mM sodium pyruvate, 100IU/m1 penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 5×10-5M2-mercaptoethanol supplemented with heat inactivated 10% human serum(Institut Jacques Boy, Reims, France). They were stimulated with eachhTERT peptide (10 μg/ml) and recombinant human IL-7 (20 ng/ml; R&DSYSTEMS®) was added.

On day 7, lymphocytes were re-activated with peptide-pulsed γ-irradiatedautologous PBMCs (50 Gy). The next day, 20 IU/ml human IL-2 (Roche,Mannheim, Germany) was added to the culture. CTL lines were re-activatedweekly during four cycles. For some donors, CD8⁺ T cells were purifiedafter three round cycle, using CD8 microbeads (Miltenyi Biotec, BergischGladbach, Germany.) according to manufacturer's recommendations andactivated once before functional test. Cytolytic assays were performed 6days after the last re-activation against various ⁵¹Cr-labeled targets:T2-B7 pulsed with tested hTERT peptides or irrelevant peptide, or tumorcell lines.

In some experiments, tumors cells were incubated with an anti-HLA classI framework mAb, w6/32 (BD Pharmigen) or anti-HLA-*B0702 mAb, ME.1, oran anti-HLA-DR mAbB, G46.6 (BD Pharmigen) at an optimal concentration(10 μg/mL) for 30 minutes to determine whether cytotoxicity wasrestricted to HLA class I.

II—Results 1. Immunogenicity of HLA-*B-0702 Predicted Peptides Derivedfrom hTERT

Using a T-cell epitope prediction program, we analysed the hTERT proteinsequence and retained six peptides (3 nonamers and 3 decamers) due totheir high predictive score (Table I). We next tested their ability tobind HLA-B0702 molecule using antigen-transporter (TAP)-deficient T2cells transfected with HLA-B0702 gene (T2-B7). Three peptides p1, p4 andp68 respectively show a high relative affinity (RA≦1) and the threeothers p277, p342 and p351 exhibited medium RA>1 (Table I). These dataindicate that these six peptides are excellent binders to HLA-B0702.Therefore, one might expect such complexes, formed in the endoplasmicreticulum, to reach the surface of tumor cells and be available for CTLrecognition.

To test if these peptides are immunogenic in vivo, we have immunizedHLA-*B0702 transgenic mice. The six peptides tested were shown to beable to induce CTL responses, although differences were noticed (TableI). Two peptides p4 and p68, binding with a high affinity to the HLA-B7molecules, induce strong CTL response in all mice tested. In contrast,peptides (p277 and p342), having lower affinity for the HLA-B7molecules, enable the generation of moderate specific CTL, in only 50%of mice tested. CTL lines specific for hTERT epitopes, generated fromtransgenic mice, recognized human T2-B7 pulsed with their respectivespecific peptides (data not shown), demonstrating the high affinity oftheir TCR for the complex MHC/peptide. Thus, there is an overallcorrelation between the results of binding/stabilization of HLAB0702 andthe in vivo CTL response in HLA-B7 transgenic mice.

TABLE 1 Immunogenicity of selected hTERT HLA-*B0702 binding peptideSEQ ID Immunogenicity‡ Peptide* Sequence NO: Score§ Relative avidity†R/T** Specific Lysis (%) p1 MPRAPRCRA 6 23 0.4 4/6 30, 39, 35, 28 p4APRCRAVRSL 7 25 0.3 6/6 52, 53, 48, 51, 46, 49 p68 APSFRQVSCL 8 25 0.46/6 63, 78, 75, 69, 70, 72 p277 RPAEEATSL 9 23 4.7 3/6 29, 33, 29 p342RPSFLLSSL 10 23 2.5 3/6 26, 39, 32, p351 RPSLTGARRL 11 23 1.5 4/647, 31, 37, 26 *The FIGURE represents the first amino acid of thepeptide. Therefore, “p4” indicates that the alanine residue is thefourth amino acid of the coding sequence. §Algorithm score obtained byusing SYFPEITHI predictive program †The relative avidity of hTERTpeptides for HLA-B*0702 was measured by using a MHC stabilization assayin comparison with a reference peptide as detailed in material andmethods. ‡HLA-B*0702 transgenic mice were immunized with candidatepeptides (six mice for each peptide) as detailed above. Ten days later,spleen cells of individual mice were restimulated in vitro with eachhTERT peptide. Cytolytic activities were assayed by 4-5h 51Cr releaseassay using peptide loaded RMA-B7 target cells. The Specific lysis wascalculated by subtracting non-specific lysis observed with the R10TVcontrol peptide. Specific lysis at a 75:1 effector/target ratio wasshowed. **R/T: responder (specific lysis ≧ 10%) versus tested mice.

2. hTERT-Derived Peptides were Processed in HLA-B0702 Transgenic Mice

To assess the presentation of endogenously synthesized hTERT peptides inthe context of the HLA-B0702 molecule against these six hTERT epitopesidentified, HLA-*B0702 transgenic mice were immunised with cDNA encodinghTERT, forty days after peptide-specific CTL responses within spleencells of individual mice were evaluated. As shown in FIG. 2, hTERTpeptide-specific CTLs were induced in most immunized mice (M), from 50to 80% of mice for p4, p68, p1, p277 and p351. In contrast,p342-specific CTLs can be induced in about 15% tested mice. Nosignificant hTERT-specific CTL responses were also induced fromnon-immunized naïve mice. Thus, these six hTERT epitopes are effectivelyintracellularly processed. Moreover, natural peptides similar in term ofamino acid sequence or structure to the synthetic ones, are presented bythe corresponding HLA-*B0702 molecules on the cell surface.

Further, these data show that multiple CTL specificity can be inducedsimultaneously against several hTERT epitopes in a single mice andvalidate our HLA-Class I transgenic mouse model for their potential totest candidate vaccines.

3. Induction of Primary CTL Responses from Healthy Donors by HTERTPeptides

We studied whether hTERT peptides would be effective in raisingHLA-B7-restricted CTLs, using PBMCs of HLA-B0702 healthy donors in an invitro immunization protocol. CTL responses were generated in eight outof ten individuals (d₁ to d₈), and peptide-specific CTL responses wereobtained in at least 50% of donors, except for p342 (20%) (FIG. 3).hHTERT epitope recognition by in vitro generated CTLs varies amongdonors, depending upon their genetic background (FIG. 3). Therefore, byrandom testing of HLA-B0702 healthy donors, it was clearly establishedthat these hTERT peptides are immunogenic in human, implying thatspecific CTL precursors for hTERT are not deleted in the peripheraladult repertoire. Therefore, we asked whether CTL lines generated fromhealthy donors would be able to kill HLA-matched hTERT⁺ tumor cells.

4. Specific hTERT CTL were Able to Lyse Tumors of Differents Origins

hTERT-specific CTLs from donors were tested for their capacity to lysehuman tumour cell lines of different origins. The results presented inTable 2 show that, CTL lines generated in vitro from healthy donorskilled HLA-B0702⁺ tumour cells, whereas no cytotoxicity againstHLA-B0702⁻ tumors was detected. (See for example CTLp351 in d₁, d₂ andd₃ in KU L268 or 293-UT target (respectively 52, 25, 20 and 34, 41 and19%) versus T1 or BBG1 target (respectively 9, 2, 6 and 0, 0, 2%).Differences were observed in tumor recognition according to the CTLspecificity; this could be explained by differential presentation ofhTERT peptides on the surface of the tumor cells. Importantly,p351-specific CTL lines generated from different donors recognize themajority of tumor cell lines tested (Table 2). In contrast, allp4-specific CTL lines do not lyse all the type of tested tumors. CTLlines, specific for p1 and p68 peptides, only recognize the T1-B7targets. p342-specific CTL lines recognize only melanoma cells (LB 34and KU 268 target). Finally, p277-specific CTL lines recognize renalcancer (293 UT) but neither melanoma nor lymphoid tumor cells. On theother hand, normal PBMCs and CD40 activated B cells were not lysed bythese hTERT peptide-specific CTL lines, regardless of HLA type (two lastlines of Table 2).

As shown in FIG. 4, the cytotoxic activity of CTLp351 line towardHLA-B7⁺ tumor cells is inhibited by an anti-class I mAb anti-HLA-B0702,but not by an anti-HLADR mAb (MHC class II). Similar data were obtainedwith other peptide-specific CTL lines (data not shown) and suggest thatCTL lines exert cytotoxicity against hTERT⁺ tumor cells in anHLA-B0702-restricted manner. Collectively, these results show that thesesix hTERT derived peptides are not equally naturally expressed at thetumor cell surface and that hTERT peptide-specific CTLs can discriminatebetween tumor cells and normal cells, through the recognition of hTERTpeptide in context of HLA-B0702 molecules.

5. Lentiviral Vector Encoding hTERT Vaccination Induces EfficientPeptide-Specific T Cell Responses in Mice

We next tested candidate vaccines, comprising either a full-length hTERTgene or a non-functional hTERT gene, inserted in a HIV-derived flapvector (FIG. 7). Previous data have shown that lentiviral vectors ofthis type target dendritic cells in vitro and in vivo, and induce strongpoly-specific anti tumor CTL responses in animals. Therefore, weimmunized HLA-*B0702 transgenic mice with either recombinant Lv-hTERT orwith pTRIP-CMV-ΔhTERT. Twelve days after, spleen cells of individualmice were evaluated by an ex vivo ELISPOT assay.

As shown in FIG. 5A, peptide-specific CD8⁺ T cell responses wereobtained against HLA-B0702 restricted hTERT epitopes, as compared withmice that received Lv-GFP control vector. Functional analysis of theinduced peptide-specific CD8⁺ cells in chromium release assay after invitro stimulation confirmed ex vivo ELISPOT data (Table 3) and show thatefficient specific CTL response is generated against these six peptidesin about 50-70% of mice after a single injection of Lv-hTERT and in 100%of the mice after a boost with TRIPLv-hTERT (FIG. 6). This was alsoassociated with strong CTL responses in all mice (Table 3).Additionally, as show in Table 3, immunization of HHD mice transgenicfor HLA-A2.1 with the same vector induced potent CTL responses specificfor two HLA-A2.1.1 restricted epitopes previously classified as dominant(p540) and cryptic (p572). Collectively, these results clearly show thatadministration of Lv-hTERT result in the induction of very efficientmulti-specific T cell response in mice, supporting that hTERT couldserve as polyepitope and polyallelic TAA for cancer immunotherapy.

As shown in FIG. 8, hTERT peptide-specific CD8⁺ T cell responses weredetected ex vivo in HLA-A2 transgenic (Tg) mice after a single injectionof recombinant pTRIP-CMV-ΔhTERT. We showed that CD8⁺ T cells specificfor p540 and PY572 epitopes were induced at least in 50% of immunizedmice (FIG. 8). These results clearly showed that the two epitopes werecorrectly endogenously processed and presented in HLA-A2 Tg mice afterimmunization with pTRIP-CMV-ΔhTERT.

Collectively, these results showed that a single injection of TRIP-hTERTresulted in the induction of a potent multi-specific anti-hTERT CD8⁺T-cells response in both HLA transgenic mice groups.

TABLE 2 anti-hTRT CTL from normal donors lyses tumor cells of varioustypes Percent lysis* CTL p1 CTL p4 CTL p68 CTL p277 CTL p342 CTL p351Cell target Cell type HLA-B7 d 1 d 6 d 8 d 1 d 6 d 8 d 1 d 6 d 8 d 1 d 6d 8 d 1 d 6 d 8 d 1 d 6 d 8 T1 T-B hybrid − 9 10 5 8 6 nd 0 7 6 3 5 9 36 4 9 2 6 T1-B7 T-B hybrid + 29 26 14 5 7 nd 19 30 4 56 30 12 2 17 5 3824 31 Sk23mel Melanoma − 0 1 0 0 2 0 10 3 4 1 4 0 4 4 3 5 2 1 LB34Melanoma + 5 4 0 0 4 2 13 7 11 0 0 2 28 22 14 52 25 20 KUL68 Melanoma +9 4 7 6 3 0 0 nd 0 7 4 nd 26 14 9 34 41 19 293-UT Renal cell + 2 8 4 1 67 3 0 0 36 17 22 9 17 4 28 22 25 BBG1 EBV-B cell − 0 4 3 0 0 2 2 0 1 0 05 1 1 0 0 0 2 JY EBV-B cell + 2 0 4 0 6 nd 0 0 9 3 0 1 8 10 6 27 18 15Raji B lymphoma − 4 1 nd 5 0 nd 0 0 nd 6 nd 1 0 nd 0 4 0 nd AutologousPBMC Normal cell + 0 0 0 4 0 1 0 0 0 0 0 1 0 2 0 0 0 1 Autologous B cellCD40^(§) Normal cell + 0 0 0 3 0 2 2 0 0 0 0 6 0 2 3 0 0 4 *hTERTpeptide specific-CTL lines (CTLp1, CTLp4, CTLp68, CTLp277, CTLp342,CTLp351) were obtained from healthy donors that were responder aftersubsequent in vitro immunization as described in material and methods.Cytotoxicity was measured in a standard ⁵¹Cr-labeled release assay.Specific lysis: for a 30:1 effector:target ratio were shown^(§)Autologous B lymphocytes from normal donors were activated for 48 hwith a trimeric CD40 L (40 μg/ml).

TABLE 3 Induction of CTL responses following Lv-hTERT immunization Flap+Lv-hTRT immunization HLA-B7 Tg mice HHD mice Restimulating Restimulatingpeptide R/T Specific lysis (%) peptide R/T Specific lysis (%) p1 4/8 27,30, 29, 32 P540 2/6 21, 18 p4 6/8 18, 25, 54, 33, 16 pY572 5/6 22, 19,14, 35, 24 p68 4/8 15, 64, 24, 16, p277 5/8 21, 25, 23, 52, 33 p342 4/818, 24, 20, 37 p351 5/8 17, 20, 18, 36, 19

III—Conclusion

New hTERT epitopes, which are in vivo immunogenic and processed inH-2-class I knockout HLA-B0702 transgenic mice have identified. Further,in vitro, hTERT peptide immunization using HLA-B702⁺ PBL from healthydonors induce specific CTL responses recognizing hTERT⁺ tumors fromvarious origins, implying that there is no deletion in the human T cellrepertoire for these epitopes. Moreover, it was shown that dependingupon the tumor origins, peptides repertoire expressed on the cellsurface could be qualitatively different, underlining, the utility tocharacterize hTERT as polyepitope tumor associated antigens forcircumvent antigenic variability of cancer cells. Finally, a humanizedHLA-*B0702 and HLA-A2 1 transgenic mice were used, to test a candidatevaccine consisting of a non-functional telomerase gene inserted in a newgeneration of lentiviral derived flap vector. A strong hTERT specificCD8⁺ T cell responses were observed in all the HLA-transgenic mice.These data support the use for therapeutic vaccination in cancerpatients and extend the potential applicability of hTERT as atherapeutic target to cover a large population of cancer patients.

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The invention claimed is:
 1. A method of inducing an HLA-B7-restrictedimmune response against at least one hTERT epitope, comprising:administering to an animal a lentiviral vector comprising apolynucleotide sequence that encodes a modified hTERT protein comprisingthe amino acid sequence of SEQ ID NO: 39; expressing the modified hTERTprotein; and inducing an HLA-B7-restricted immune response against atleast one hTERT epitope selected from: SEQ ID NO: 6 a. MPRAPRCRA  (p1),SEQ ID NO: 7 b. APRCRAVRSL (p4), SEQ ID NO: 8 c. APSFRQVSCL (p68),SEQ ID NO: 9 d. RPAEEATSL  (p277), SEQ ID NO: 10 e. RPSFLLSSL  (p342),SEQ ID NO: 11 f. RPSLTGARRL (p351).


2. The method of claim 1, wherein the lentiviral vector comprising thepolynucleotide sequence that encodes a modified hTERT protein is thepTRIP-CMV-ΔhTERT vector deposited at the CNCM under the number 1-3660 onJul. 28,
 2006. 3. The method of claim 1, further comprising providingantigen presenting cells (APCs), transforming the APCs in vitro with thelentiviral vector comprising the polynucleotide sequence that encodes amodified hTERT protein, and administering the APCs to the animal.
 4. Themethod of claim 3, wherein the APCs are dendritic cells.
 5. The methodof claim 1, wherein the animal is a human.
 6. The method of claim 1, themethod comprising inducing an HLA-B7-restricted immune response againstat least two hTERT epitopes selected from: SEQ ID NO: 6a. MPRAPRCRA  (p1), SEQ ID NO: 7 b. APRCRAVRSL (p4), SEQ ID NO: 8c. APSFRQVSCL (p68), SEQ ID NO: 9 d. RPAEEATSL  (p277), SEQ ID NO: 10e. RPSFLLSSL  (p342), and SEQ ID NO: 11 f. RPSLTGARRL (p351).


7. The method of claim 6, wherein the lentiviral vector comprising thepolynucleotide sequence that encodes a modified hTERT protein is thepTRIP-CMV-ΔhTERT vector deposited at the CNCM under the number 1-3660 onJul. 28,
 2006. 8. The method of claim 6, further comprising providingantigen presenting cells (APCs), transforming the APCs in vitro with thelentiviral vector comprising the polynucleotide sequence that encodes amodified hTERT protein, and administering the APCs to the animal.
 9. Themethod of claim 8, wherein the APCs are dendritic cells.
 10. The methodof claim 6, wherein the animal is a human.
 11. The method of claim 1,the method comprising inducing an HLA-B7-restricted immune responseagainst each of hTERT epitopes: SEQ ID NO: 6 a. MPRAPRCRA  (p1),SEQ ID NO: 7 b. APRCRAVRSL (p4), SEQ ID NO: 8 c. APSFRQVSCL (p68),SEQ ID NO: 9 d. RPAEEATSL  (p277), SEQ ID NO: 10 e. RPSFLLSSL  (p342),and SEQ ID NO: 11 f. RPSLTGARRL (p351).


12. The method of claim 11, wherein the lentiviral vector comprising thepolynucleotide sequence that encodes a modified hTERT protein is thepTRIP-CMV-ΔhTERT vector deposited at the CNCM under the number 1-3660 onJul. 28,
 2006. 13. The method of claim 11, further comprising providingantigen presenting cells (APCs), transforming the APCs in vitro with thelentiviral vector comprising the polynucleotide sequence that encodes amodified hTERT protein, and administering the APCs to the animal. 14.The method of claim 13, wherein the APCs are dendritic cells.
 15. Themethod of claim 11, wherein the animal is a human.